Novel human phosphodiesterase IV isozymes

ABSTRACT

This invention relates to novel nucleic acid sequences encoding three novel human phosphodiesterase (hPDE IV) isozymes. It also relates to polypeptides encoded by such sequences.  
     This invention also relates to an assay method for detecting the presence of such novel isozymes in human cells, and to a method of identifying compounds or other substances that inhibit or modify the activity of such isozymes.

BACKGROUND OF THE INVENTION

[0001] This invention relates to novel nucleic acid sequences encodingthree novel human phosphodiesterase IV (hPDE IV) isozymes.

[0002] Cyclic nucleotide phosphodiesterases (PDE's) are a family ofenzymes that catalyze the degradation of cyclic nucleotides. Cyclicnucleotides, particularly cAMP, are important intracellular secondmessengers, and PDEs are one cellular component that regulates theirconcentration. In recent years, five PDE enzymes (PDE I-PDE V), as wellas many subtypes of these enzymes, have been defined based on substrateaffinity and cofactor requirements (Beavo J A and Reifsnyder D H, TrendsPharmacol. Sci. 11:150 [1990]; Beavo J, in: Cyclic NucleotidePhosphodiesterases: Structure, Regulation and Drug Action. Beavo J andHousley M D (Eds.). Wiley: Chichester, pp. 3-15 [1990]).

[0003] Theophylline, a general PDE inhibitor, has been widely used inthe treatment of asthma. It has been speculated that selectiveinhibitors of PDE isozymes and their subtypes (particularly thecAMP-specific PDE IV) will lead to more effective therapy with fewerside effects (for reviews, see Wieshaar R E et al., J. Med. Chem.,28:537 [1985] and Giembycz M A, Biochem. Pharm., 43:2041 [1992], Lowe JA and Cheng J B, Drugs of the Future, 17:799-807 [1992]). However, evenPDE IV selective drugs such as rolipram suffer from emetic side effectsthat limit their use. An even more selective approach is to inhibitindividual subtypes of PDE IV, each one of which is expected to have itsown tissue distribution. If the PDE IV isozyme responsible for efficacyis different from that causing side effects, an isozyme selective drugcould separate therapeutic and side effects. The cloning and expressionof the human PDE IVs would greatly aid the discovery of isozymeselectiveinhibitors by providing purified isoenzymes to incorporate into drugassays.

[0004] Mammalian PDE IV, the homologue of the Drosophila Dunce gene(Chen C N et al., Proc. Nat. Acad. Sci. (USA) 83:9313 [1986]), is knownto have four isoforms in the rat (Swinnen J V et al., Proc. Nat. Acad.Sci. (USA) 86:5325 [1989]). The cloning of one human isoform of PDE IVfrom monocytes was reported in 1990 (Livi G P et al., Mol. Cell. Bio.,10:2678 [1990]). From Southern blot data, the authors concluded thatthis enzyme was probably the only PDE IV gene in humans, with thepossible exception of one other isozyme. The same group has recentlypublished the sequence of a second human isoform isolated from brainthat they designate hPDE IV-B to distinguish it from the monocyte form,which they designate as hPDE IV-A (McLaughlin M M et al., J. Biol. Chem.268:6470 [1993]). For clarity, we will use this nomenclature as well.

[0005] Our invention relates to the nucleic acid sequences encodingthree novel human PDE IV isozymes generated by differential splicingfrom a single gene. We designate these isoforms as hPDE IV-B1, hPDEIV-B2 and hPDE IV-B3. The hPDE IV-B2 sequence encodes a polypeptidenearly identical to that reported for hPDE IV-B (McLaughlin M M et al.,J. Biol. Chem. 268:6470 [1993]), and the hPDE IV-B2 splice variantrepresents the unspliced genomic sequence with respect to thedifferential splice site. Of the two other splice variants, hPDE IV-B1encodes the longest polypeptide chain, as well as the N-terminalsequence homologous to its rat homologue, DPD (Colicelli J, et al.,Proc. Nat. Acad. Sci. (USA) 86:3599 [1989]).

[0006] The novel human PDE IV DNA sequences and their encoded peptidesmay be used to screen for drugs that are selective for a particularhuman PDE IV isozyme. Such novel DNA sequences may also be used inassays to detect the presence of a particular PDE IV isozyme in humancell lines, thus providing information regarding the tissue distributionof each isozyme and its biological relevance with respect to particulardisease states.

[0007] The following abbreviations are used throughout this patent: BALbronchoalveolar lavage bp base pair(s) cAMP cyclic adenosine3′,5′-monophosphate dNTP 2′-deoxynucleoside-5′-triphosphate dATP2′-deoxyadenosine-5′-triphosphate dCTP 2′-deoxycytidine-5′-triphosphatedGTP 2′-deoxyguanidine-5′-triphosphate dTTP2′-deoxythymidine-5′-triphosphate hPDE IV-A human monocyte PDE IV hPDEIV-B human brain PDE IV hPDE IV-B1 human brain PDE IV, splice variant 1hPDE IV-B2 human brain PDE IV, splice variant 2 hPDE IV-B3 human brainPDE IV, splice variant 3 kb kilobase(s) PCR polymerase chain reactionPDE cyclic nucleotide phosphodiesterase PDE I Ca²⁺/Calmodulin-dependentPDE PDE II cGMP stimulated PDE PDE III cGMP inhibited PDE PDE IV highaffinity cAMP-specific PDE PDE V cGMP specific PDE RACE RapidAmplification of cDNA Ends RT avian myeloblastosis virus (AMV) reversetranscriptase RT-PCR PCR of RT-transcribed mRNA SSC 1 × SSC = 0.15 MNaCl, 0.015 Na₃ citrate pH 7.0

[0008] The nucleotides and amino acids represented in the varioussequences contained herein have their usual single letter designationsused routinely in the art.

SUMMARY OF THE INVENTION

[0009] This invention relates to novel nucleic acid sequences encodingthe novel hPDE IV isozymes hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. Morespecifically, it relates to DNA segments comprising, respectively, theDNA sequences of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 and SEQUENCE IDNO. 3, as defined below, or alleleic variations of such sequences. Italso relates to polypeptides produced by expression in a host cell intowhich has been incorporated one of the foregoing DNA sequences or analleleic variation of such sequence.

[0010] This invention also relates to an isolated polypeptide comprisingthe amino acid sequence of SEQUENCE ID NO. 4, SEQUENCE ID NO. 5 orSEQUENCE ID NO. 6.

[0011] This invention also relates to recombinant DNA comprising the DNAsequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3,or an alleleic variations of such sequence.

[0012] This invention also relates to an isolated DNA segment comprisingthe genomic promoter region that regulates transcription or translationof the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCEID NO. 3, or an alleleic variation of such sequence.

[0013] This invention also relates to an assay method for detecting thepresence of hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3 in human cells,comprising: (a) performing a reverse transcriptase-polymerase chainreaction on total RNA from such cells using a pair of polymerase chainreaction primers that are specific for, respectively, hPDE IV-B1, hPDEIV-B2 or hPDE IV-B3, as determined from, respectively: (i) the DNAsequence of SEQUENCE ID NO. 1 or an alleleic variation thereof; (ii) theDNA sequence of SEQUENCE ID NO. 2 or an alleleic variation thereof; or(iii) the DNA sequence of SEQUENCE ID NO. 3 or an alleleic variationthereof, and (b) assaying the appearance of an appropriately sized PCRfragment by agarose gel electrophoresis.

[0014] This invention also relates to a method of identifying compoundsor other substances that inhibit or modify the activity of hPDE IV-B1,hPDE IV-B2 or hPDE IV-B3, comprising measuring the activity of,respectively, hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, in: (a) a cell lineinto which has been incorporated recombinant DNA comprising the DNAsequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3,or an alleleic variation thereof, or (b) a cell line that naturallyselectively expresses hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, asdetermined by the assay method described above.

[0015] This invention also relates to an isolated DNA segment comprisinga DNA sequence that is a subset of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2or SEQUENCE ID NO. 3, or an alleleic variation thereof, and that iscapable of hybridizing to, respectively, SEQUENCE ID NO. 1, SEQUENCE IDNO. 2 or SEQUENCE ID NO. 3, or an alleleic variation thereof, when usedas a probe, or of amplifying all or part of such sequence when used as apolymerase chain reaction primer.

[0016] As used herein, the term “functionally equivalent DNA segment”refers to a DNA segment that encodes a polypeptide having an activitythat is substantially the same as the activity of the polypeptideencoded by the DNA to which such segment is said to be functionallyequivalent.

[0017] As used herein, the term “subset of a DNA sequence” refers to anucleotide sequence that is contained in and represents part, but notall of such DNA sequence, and is sufficient to render it specific tosuch sequence when used as a PCR primer and to render it capable ofhybridizing to such sequence when used as a probe at high stringency.

[0018] The term “functionally equivalent polypeptide” refers to apolypeptide that has substantially the same activity as the polypeptideto which it is said to be functionally equivalent.

[0019] The term “subset of a polypeptide” refers to a peptide sequencethat is contained in and represents part, but not all of suchpolypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1. hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3 Restriction Map andClone Diagram. This figure shows the relationship between the cDNAsequences encoding the three splice variants. Black boxes indicateprotein coding regions and open boxes indicate untranslated regions.

[0021]FIG. 2. hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3 DNA and TranslatedAmino Acid Sequences. (+) Numbering begins with the “A” of the ATG startcodon in hPDE IV-B3. Four stop codons are designated by “***”. Theseinclude the protein translation stop (1,552), and the stop codons thatprevent the coding region from continuing further in the 5′ direction ineach splice variant: hPDE IV-B1 (−630), hPDE IV-B2 (−270) and hPDE IV-B3(−89). The alternate splice junction is between nucleotides −23 and −24,and the putative splice acceptor sequence in hPDE IV-82 (−33 to −24) isunderlined.

[0022]FIG. 3. Alternative Splice Junction. This figure is a close-upview of the splice junction between −24 and −23, showing the threealigned sequences hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. The putativesplice acceptor sequence in hPDE IV-B2 (−33 to −24) is underlined.

[0023]FIG. 4. Amino Acid Sequence Comparison: hPDE IV-B1, hPDE IV-B2,hPDE IV-B3. and Rat DPD. Identity with the hPDE IV-B1 sequence isindicated with a dash. A translation of the region upstream of the hPDEIV-B3 start codon is shown in parenthesis to highlight the completesequence divergence of hPDE IV-B2 and hPDE IV-B3 from hPDE IV-B1 atamino acid 196.

[0024]FIG. 5. Restriction MaR of the hPDE IV-B Genomic Locus.Transcriptional orientation (5′-3′) of hPDE IV-B is from left to right,with the approximate positions of exons known by partial sequenceanalysis indicated by solid boxes (coding). The position of the stopcodon is indicated by an asterisk, followed by a segment of a 3′untranslated region (open box). Regions hybridizing strongly to the 308bp probe, as described in the text, are indicated by a dark hatched box,while weakly hybridizing regions are shown as lighter hatched regions.It is because of weak hybridization between the EcoRI and HindIII sitesin λ11.1 that we position an exon (with a “?”) in that interval. Thehybridizing restriction fragments seen in genomic blots with the 308 bpprobe are indicated below the figure.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The procedures by which the DNA sequences encoding for novelisozymes hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3 were identified andisolated as described below.

[0026] Discovery of PDE IV-B Using Degenerate PCR: The degenerate PCRprimers (5′-Deg and 3′-Deg, as described below in the section labelledMaterials and Methods) were designed against amino acid sequences thatwere conserved (with one exception) between the six published PDE IVsequences from human, rat, and Drosophila (Livi G P et al., Mol. Cell.Biol. 10:2678 [1990], Swinnen J V et al., Proc. Nat. Acad. Sci. (USA)86:5325 [1989], and Chen C N et al.. Proc. Nat. Acad. Sci. (USA) 83:9313[1986]). These primers were expected to amplify 308 bp of PDE IVsequence from any human isoform mRNA that also conserved those aminoacids. The RT-PCR was done on human BAL sample total RNA as describedbelow in the section labelled Materials and Methods, and a fragment ofthe correct size was obtained. Sequence analysis of this fragment showedit to be different from hPDE IV-A (Livi G R et al., [1990]). Thisfragment of hPDE IV-B corresponds to bp 1,575 to 1,882 in SEQUENCE IDNO. 1. This fragment was isolated from several independent PCR reactionsand sequenced to confirm that no apparent differences were due to PCRartifacts.

[0027] Isolation of a cDNA Clone for hPDE IV-B: The human medulla cDNAlibrary was screened as described below in the section labelled inMaterials and Methods, and a single cDNA clone was obtained. The insertsequence corresponds to bp 924 to 2,554 of SEQUENCE ID NO. 1, and wasclearly not full length in the coding region by comparison with theknown PDE IV sequences. Also, since no polyA tract was found at the 3′end of this clone, we do not believe that the 3′ untranslated region iscomplete; however, this is of no functional significance with respect toproducing a hPDE protein. There was one nucleotide difference betweenthe cDNA sequence and the PCR fragment sequence. SEQUENCE ID NO. 1contains a C at bp 1792, the nucleotide seen in the cDNA sequence,rather than the T that has been seen at this position in PCR isolations.We believe that this difference, which changes an amino acid, is real,and represents an alleleic difference in the human population.

[0028] Completion of the cDNA Sequence using the RACE Method: The RACEmethod showed that there was not just a single 5′ end to the hPDE IV-BcDNA, but at least three. Fragments of different sizes were obtained,all beginning at the GSi oligonucleotide primer site and extendingtowards the 5′ end of the cDNA. The three fragments that weresuccessfully sequenced had a variable length of non-homologous sequenceat the 5′ end that joins the hPDE IV sequence at the same point in allthree cases. These different 5′ ends, when joined to the rest of thecDNA sequence, make three forms of the hPDE IV-B gene that we designatehPDE IV-B1 (SEQUENCE ID NO. 1), hPDE IV-B2 (SEQUENCE ID NO. 2), and hPDEIV-B3 (SEQUENCE ID NO. 3). The three hPDE IV-B isoforms makepolypeptides of different lengths. From the cDNA sequences, hPDE IV-B1is predicted to encode a protein of 721 amino acids (SEQUENCE ID NO. 4),hPDE IV-32 a protein of 564 amino acids (SEQUENCE ID NO. 5), and hPDEIV-B3 a protein of 517 amino acids (SEQUENCE ID NO. 6). The threeisoforms are shown diagrammatically in FIG. 1, and the DNA sequence andamino acid translation of the three isoforms of hPDE IV-B is shown inFIG. 2.

[0029] The most logical explanation for the three hPDE IV-B isoforms isthat they are If generated by alternative splicing of 5′ exons onto theshared 3′ sequence. The putative alternative splice junction is shown at−23 bp in FIG. 2. To test this hypothesis, we amplified PCR fragmentsfrom human genomic DNA using primers on either side of the putativesplice junction. hPDE IV-B1 and hPDE IV-B3 specific 5′ primers did notgive amplified fragments, indicating that the sequences on either sideof the putative splice lie further than 2 kb apart in genomic DNA (thepractical limit for PCR amplification). Primers specific for the hPDEIV-B2 isoform gave the identically sized fragment as predicted from thecDNA (data not shown), indicating that at least with respect to theputative splice junction at −23 bp, this is the unspliced genomicsequence. Indeed, examination of the sequence of hPDE IV-B2 at thislocation (underlined bp −33 to −24 in FIGS. 2 and 3) reveals anexcellent match for a splice acceptor sequence (Breathnach R and ChambonP, Ann. Rev. Biochem. 50:349 [1981]).

[0030] hPDE IV-B is very similar to one of the known rat isozymes, DPD(Colicelli J. et al., Proc. Nat. Acad. Sci. (USA) 86:3599 [1989]), with96.3% amino acid identity in the regions that can be aligned, ascompared to only a 74.6% identity with hPDE IV-A. However, of the threesplice variants, only hPDE IV-B1 continues to have homology to rat DPD5′ of the putative splice junction (FIG. 4). Indeed, hPDE IV-B1 extendsmuch further 5′ than rat DPD, and the homology between the two continuesto the 5′ end of rat DPD. The fact that the hPDE IV-B1 sequence has beenconserved in evolution is strong evidence that this sequence isfunctional and is translated into protein in vivo. We cannot be surethat the other two splice variants are functional in vivo, although therecent paper (McLaughlin M M et al., J. Biol. Chem. 268:6470 [1993])reporting the hPDE IV-B2 sequence has shown by expression cloning thatthis isoform can produce enzymatically active protein in a yeastexpression system.

[0031] Mammalian Expression Clones for hPDE IV-B1, B-2, -B3: The hPDEIV-B1, -B2, -B3 cDNA sequences were subcloned into the mammalianexpression vector pcDNA1-amp, a vector that is suitable for transientlyexpressing these genes in COS cells and that was constructed byreplacing the 950 bp NheI fragment of pcDNA1 (Invitrogen) with a 1.2 kbPCR fragment from pUC18 (Sigma) containing the Amp resistance gene. Theresulting expression clones are designated pc-hPDE IV-B1, pc-hPDE IV-B2,and pc-hPDE IV-B3. All three clones have been shown to direct theexpression of proteins that catalyze the degradation of cAMP whentransiently transfected into COS cells.

[0032] Genomic Sequences for hPDE IV-B: Overlapping genomic clonesdefine “26 kb of genomic sequence encoding at least the 3′ half of thehPDE IV-B gene (FIG. 5). Limited DNA sequencing of these genomic clonesconfirms that the SaII restriction site in clone λK2.1 is contained inan exon, and corresponds to the unique SaII site seen in the cDNAsequence (1,235-1,240 in Sequence ID No. 1). Hybridization data (FIG. 5)defines the orientation of the gene, and confirms the hybridizingfragment sizes seen in genomic Southern blots hybridized at highstringency with the 308 bp PCR fragment (bp 1,575-1,882 in SEQUENCE IDNO. 1) from hPDE IV-B: EcoRI6.6 kb, HindIII-4.4 kb, BamHI-4.2 kb.

[0033] Deposits

[0034] Three cDNA clones (pc-hPDE IV-B1, pc-hPDE IV-B2, and pc-hPDEIV-B3) are being deposited with the American Type Culture Collection,Rockville, Md. U.S.A. (ATCC).

[0035] Assays

[0036] Using the DNA sequence of hPDE IV-B and hPDE IV-A, one could makea large number of isoenzyme specific PCR primer pairs. We have made andtested the following hPDE IV-B and hPDE IV-A specific primer pairs. Thesequences 5′B(5′-CGAAGAAAGTTACAAGTTC-3′) and3′B(5′-AACCTGGGATTTTTCCACA-3′) are a pair of 19-mer primers thatspecifically amplify a 245 bp fragment from hPDE IV-B, and the sequences5′A(5′-CACCTGCATCATGTACATG-3′) and 3′A(5′-TCCCGGTTGTCCTCCAAAG-3′) are19-mers that amplify an 850 bp fragment specifically from hPDE IV-A. Inaddition, one skilled in the art could easily design a pair of PCRprimers specific for each of the hPDE IV-B splice variants by using theunique 5′ sequences. Using these primers, one can sensitively assay thepresence of these isozymes in any tissue from which total RNA can beisolated (e.g., by the method of Chomcynski P and N Sacchi, Anal.Biochem. 162:156 1987) by performing an RT-PCR reaction on such totalRNA using the specific primers and then assaying the amount of theappropriately sized DNA PCR product by agarose gel electrophoresis. TheRT-PCR conditions are identical to those described in Materials andMethods, except that the thermocycling parameters are as follows:Denature-94° C., 30 sec; Anneal-55° C. 30 sec; Polymerize-72° C., 60.Amplify for at least 30 cycles.

[0037] The claimed DNA sequences of this invention can be reproduced byone skilled in the art by either PCR amplification of the coding regionusing PCR primers designed from the sequences or by obtaining thedescribed cDNA clones from ATCC directly.

[0038] Utility of the Invention

[0039] A general utility of the novel human PDE IV genes and theirencoded peptides is to allow screening for human PDE IV isozymespecific/selective drugs that may be improved therapeutics in the areasof asthma and inflammation. The cloned genes make it possible, byexpression cloning methods familiar to those skilled in the art, toproduce active, purified isoenzymes that can be used in PDE IV activityassays (e.g., Davis C W, and Daly J W, J. Cyclic Nucleotide Res. 5:65[1979], Torphy T J and Cielinski L B, Mol. Pharm. 37:206 [1990]) tomeasure the potency of inhibitors against individual isoenzymes. This istrue both for distinguishing hPDE IV-A and hPDE IV-B selectiveinhibitors and for distinguishing inhibitors selective between hPDEIV-B1, hPDE IV-B2, or hPDE IV-B3. Since the hPDE IV-B splice variantsmay each have their own tissue distribution and may be pharmacologicallyseparable from each other, it may be valuable to screen for inhibitorsspecific for individual splice variants.

[0040] Genomic sequences are also of utility in the context of drugdiscovery. It may be valuable to inhibit the mRNA transcription of aparticular isoform rather than to inhibit its translated protein. Thisis particularly true with hPDE IV-B, since the different splice variantsmay be transcribed from different promoters. There is precedent formultiple promoters directing the transcription of a mouse brain2′,3′-cyclic-nucleotide 3′ phosphodiesterase (Kurihara T et al.,Biochem. Biophys. Res. Comm. 170:1074 [1990]). This invention wouldprovide the means for one skilled in the art to locate multiplepromoters. Isolation of genomic clones containing the promoter(s) andthe 5′-most exons of hPDE IV-B1, hPDE IV-B2, and hPDE IV-B3 may beaccomplished by screening a human genomic library with the unique 5′sequences. Such promoters could then be linked to a convenient reportergene such as firefly luciferase (de Wet J R et al., Mol. Cell. Biol.7:725 [1987]), transfected into a mammalian cell line, and used toscreen for agents that inhibit the activity of the promoter of interestwhile having minimal effect on other promoters.

[0041] Another utility of the invention is that the DNA sequences, onceknown, give the information needed to design assays to specificallydetect each isoenzyme or splice variant. Isozyme-specific PCR primerpairs are but one example of an assay that depends completely on theknowledge of the specific DNA sequence of the isozyme or splice variant.Such an assay allows detection of mRNA for the isozyme to access thetissue distribution and biological relevance of each isozyme to aparticular disease state. It also allows identification of cell linesthat may naturally express only one isozyme—a discovery that mightobviate the need to express recombinant genes. If specific hPDE IVisozymes are shown to associated with a particular disease state, theinvention would be valuable in the design of diagnostic assays to detectthe presence of isozyme mRNA.

[0042] Materials and Methods

[0043] (a) Cells/Reagents

[0044] Cells from a human bronchoalveolar lavage (BAL) were purchasedfrom the Johns Hopkins University (Dr. M. Uu). Human brainstem tissuewas purchased from the International Institute for the Advancement ofMedicine. Unless noted below, all restriction endonucleases and DNAmodifying enzymes were from Boehringer-Mannheim.

[0045] (b) Degenerate RT-PCR

[0046] Total RNA was isolated from human tissue as previously described(Chomcynski P and Sacchi N, Anal. Biochem. 162:156 [1987]). To preparean 80 μl reverse transcriptase (RT) reaction, 4 μg total RNA and 4 μgrandom hexamer primers (Pharmacia/LKB) were heated to 90° C. for 5 minin 60 μl RNase free water. After chilling on ice, the reaction wasbrought to 80 μl and the following conditions by the addition ofconcentrated stocks: 1× RT buffer (50 mM Tris pH 8.3, 6 mM magnesiumchloride, 40 mM KCl); 1 mM each dATP, dGTP, dCTP, and dTTP; 1 mMdithiothreitol; 25 U/mi RNasin (Promega); and 900 U/ml AMV reversetranscriptase (RT). Incubate at 42° C. for 1 hour, then boil for 5minutes to inactivate the RT.

[0047] A 50 μl PCR reaction was set up by using 3.25 μl of the abovereaction mix. Final buffer conditions were (including carryover fromRT): 10 mM Tris pH 8.3, 50 mM potassium chloride, 1.5 μM magnesiumchloride, 10 μg/ml bovine serum albumin, 2.5% (v/v) Formamide, 200 μMeach dNTP, 0.5 pmol/μl each degenerate primer(5′-Deg=5′-CAGGATCCAAPACNATGGTNGAPAC-3′,3′-Deg=5′-GCTCTAGATCNGCCCANGTYTCCCA-3′, where N=A, G, C, or T, P=A or Gand Y=C or T) and 0.05 U/μl Amplitaq polymerase (Perkin Elmer).Amplification was done in a Perkin Elmer 9600 PCR thermocycler using thefollowing parameters: denature—94° C., 30 sec; anneal-37° C.+0.5°C./cycle, 60 sec+1 sec/cycle; polymerize—72° C., 60 sec. Amplify for 35cycles.

[0048] (c) Library Screening

[0049] 8×10⁵ clones from a commercially available human medulla cDNAlibrary (Clontech # HL 1089a) were screened with an 857 bp DNA fragmentcontaining the entire conserved catalytic domain of hPDE IV-A. Thisfragment was generated by RT-PCR amplification from the Jurkat T-cellline mRNA using unique primers to amplify bp 573-1430 from the PDE IV-Asequence (Livi G P, et al., Mol. Cell. Bio., 10:2678 [1990]). Thefragment was labeled to a specific activity>5×10⁸ cpm/μg, and hybridizedunder the following conditions: 6× SSC, 5× Denhardt's Solution (1×Denhardt's=0.02% each of Ficoll, polyvinylpyrrolidone, and bovine serumalbumin), 0.1% sodium dodecyl sulfate (SDS), 100 μg/ml yeast tRNA. Probeconcentration was 4×10⁵ cpm/ml. Filters were hybridized at 65° C.for >16 hours, and then washed to a final stringency of 1× SSC at 55° C.

[0050] 1×10⁶ clones from a commercially available human genomic library(Clontech #HL1111j) were screened with the 308 bp PCR fragment of hPDEIV-B (bp 1,575 to 1,882 in SEQUENCE ID NO. 1) and the homologousfragment from hPDE IV-A. The screening conditions were as follows: 5×SSC, 5× Denhardts solution (see above), 40% formamide, 0.5% sodiumdodecyl sulfate, and 20 μg/ml herring sperm DNA. Probe concentration was4×10⁵ cpm/ml. The filters were hybridized at 42° C. for >16 hours, andthen washed to a final stringency of 0.5× SSC at room temperature. Agenomic library was also constructed in the vector LambdaGEM12 (Promega)using the XhoI half-site method, and 1×10⁸ clones screened under thesame hybridization conditions used for the previous genomic library.

[0051] (d) DNA Sequencing

[0052] All DNA sequencing was done using an ABI model 373A DNA sequenceron DNA fragments cloned into various pGEM vectors (Promega). Sequencingreactions were done using the Taq sequencing method.

[0053] (e) RACE Method

[0054] The RACE method (Rapid Amplification of cDNA Ends) was adaptedfrom a published method (Frohman M A and Martin G R, In: Technique—aJournal of Methods in Cell and Molecular Biology, Vol. 1, No. 3, pp.165-170 [1989]). In order to produce the 5′ end of the cDNA, an RTreaction was performed on human brainstem total RNA as above with theexception that the gene specific RT primer (GS-RT:5′-GCAAGTTCTGAATTTGT-3′) was at a concentration of 0.1 pmol/μl. Thereaction was incubated at 42° C. for 1 hour and then shifted to 52° C.for 30 min. This higher temperature seems to be critical to avoiding apremature truncation product presumably caused by a sequence that AMV RThas difficulty reading through.

[0055] After removing buffers using a Centricon 30 filtration device andconcentrating in a speedvac, one tails the cDNA with dATP using terminaltransferase (TdT) in a 20 μl reaction volume. Final conditions are: 1×TdT buffer (40 mM K-Cacodylate pH 6.8, 0.1 mM dithiothreitol), 0.75 mMCoCl₂, 0.2 mM dATP, 1,250 U TdT/ml. Incubate 37° C. for 5 min,inactivate TdT at 65° C. 5 min. This reaction is diluted with water to500 μl and used as a template in a series of nested PCR reactions.

[0056] The first PCR amplification (50 ml) uses the same PCR bufferconditions as above, but uses three primers: the Primer/Adaptor(Ro-dT₁₇: 5′-AAGCATCCGTCAGCATCGGCAGGACAAC(T₁₇)-3′) at 0.2 pmol/μl, theForward Outside Primer (Ro: 5′-AAGCATCCGTCAGCATC-3′) at 0.5 pmol/μl, andthe Gene-Specific Reverse Outside Primer (GSo: 5′-ATGGCAGCCAGGATTTC-3′)at 0.5 pmol/μl. Taq DNA polymerase is only added after denaturing thereaction to 95° C. for 5 min and equilibrating to 72° C. For the firstcycle, the annealing step is 10 min at 55° C., and the extension is at72° C. for 40 min. After that, cycling parameters (PE 9600 machine) are:Denature 94° C., 30 sec; Anneal 53° C., 30 sec; Polymerize 72° C., 45sec. Amplify 28 cycles. Dilute this product 20× to serve as template fora second PCR reaction using primers nested just inside those used in thefirst PCR reaction. This greatly increases the specificity of the finalPCR products.

[0057] The second 50 μl PCR reaction uses identical buffer conditions tothe first, and uses 1 μl of the 20× diluted product from the first PCRreaction as template DNA. The primers are the Forward Inside Primer (Ri:AGCATCGGCAGGACAAC-3′) and Gene-Specific Inside Primer (GSi:5′-GGTCGACTGGGCTACAT-3′) both at 0.5 pmol/μl. For 12 cycles, theparameters are the same as the final 28 cycles of the previousamplification. The annealing temperature is then raised to 60° C. foranother 18 cycles. Products are then analyzed on an agarose gel. Theyshould extend from the GSi primer to the 5′ end of the mRNA(s).

[0058] Sequence ID Summary

[0059] 1. hPDE IV-B1 cDNA sequence. 2,554 bp.

[0060] 2. hPDE IV-B2 cDNA sequence. 2,246 bp.

[0061] 3. hPDE IV-B3 cDNA sequence. 2,045 bp.

[0062] 4. Predicted amino acid sequence of hPDE IV-B1. 721 amino acids.

[0063] 5. Predicted amino acid sequence of hPDE IV-B2. 564 amino acids.

[0064] 6. Predicted amino acid sequence of hPDE IV-B3. 517 amino acids.

1 6 2554 base pairs nucleic acid double linear cDNA 1 TGGATGGTGAAAGCTAGCAC TCCTTACAAG ATATGACAGC AAAAGATTCT TCAAAGGAAC 60 TTACTGCTTCTGAACCTGAG GTTTGCATAA AGACTTTCAA GGAGCAAATG CATTTAGAAC 120 TTGAGCTTCCGAGATTACCA GGAAACAGAC CTACATCTCC TAAAATTTCT CCACGCAGTT 180 CACCAAGGAACTCACCATGC TTTTTCAGAA AGTTGCTGGT GAATAAAAGC ATTCGGCAGC 240 GTCGTCGCTTCACTGTGGCT CATACATGCT TTGATGTGGA AAATGGCCCT TCCCCAGGTC 300 GGAGTCCACTGGATCCCCAG GCCAGCTCTT CCGCTGGGCT GGTACTTCAC GCCACCTTTC 360 CTGGGCACAGCCAGCGCAGA GAGTCATTTC TCTACAGATC AGACAGCGAC TATGACTTGT 420 CACCAAAGGCGATGTCGAGA AACTCTTCTC TTCCAAGCGA GCAACACGGC GATGACTTGA 480 TTGTAACTCCTTTTGCCCAG GTCCTTGCCA GCTTGCGAAG TGTGAGAAAC AACTTCACTA 540 TACTGACAAACCTTCATGGT ACATCTAACA AGAGGTCCCC AGCTGCTAGT CAGCCTCCTG 600 TCTCCAGAGTCAACCCACAA GAAGAATCTT ATCAAAAATT AGCAATGGAA ACGCTGGAGG 660 AATTAGACTGGTGTTTAGAC CAGCTAGAGA CCATACAGAC CTACCGGTCT GTCAGTGAGA 720 TGGCTTCTAACAAGTTCAAA AGAATGCTGA ACCGGGAGCT GACACACCTC TCAGAGATGA 780 GCCGATCAGGGAACCAGGTG TCTGAATACA TTTCAAATAC TTTCTTAGAC AAGCAGAATG 840 ATGTGGAGATCCCATCTCCT ACCCAGAAAG ACAGGGAGAA AAAGAAAAAG CAGCAGCTCA 900 TGACCCAGATAAGTGGAGTG AAGAAATTAA TGCATAGTTC AAGCCTAAAC AATACAAGCA 960 TCTCACGCTTTGGAGTCAAC ACTGAAAATG AAGATCACCT GGCCAAGGAG CTGGAAGACC 1020 TGAACAAATGGGGTCTTAAC ATCTTTAATG TGGCTGGATA TTCTCACAAT AGACCCCTAA 1080 CATGCATCATGTATGCTATA TTCCAGGAAA GAGACCTCCT AAAGACATTC AGAATCTCAT 1140 CTGACACATTTATAACCTAC ATGATGACTT TAGAAGACCA TTACCATTCT GACGTGGCAT 1200 ATCACAACAGCCTGCACGCT GCTGATGTAG CCCAGTCGAC CCATGTTCTC CTTTCTACAC 1260 CAGCATTAGACGCTGTCTTC ACAGATTTGG AAATCCTGGC TGCCATTTTT GCAGCTGCCA 1320 TCCATGACGTTGATCATCCT GGAGTCTCCA ATCAGTTTCT CATCAACACA AATTCAGAAC 1380 TTGCTTTGATGTATAATGAT GAATCTGTGT TGGAAAATCA TCACCTTGCT GTGGGTTTCA 1440 AACTGCTGCAAGGAGAACAC TGTGACATCT TCATGAATCT CACCAAGAAG CAGCGTCAGA 1500 CACTCAGGAAGATGGTTATT GACATGGTGT TAGCAACTGA TATGTCTAAA CATATGAGCC 1560 TGCTGGCAGACCTGAAGACA ATGGTAGAAA CGAAGAAAGT TACAAGTTCA GGCGTTCTTC 1620 TCCTAGACAACTATACCGAT CGCATTCAGG TCCTTCGCAA CATGGTACAC TGTGCAGACC 1680 TGAGCAACCCCACCAAGTCC TTGGAATTGT ATCGGCAATG GACAGACCGC CTCATGGAGG 1740 AATTTTTCCAGCAGGGAGAC AAAGAGCGGG AGAGGGGAAT GGAAATTAGC CCAATGTGTG 1800 ATAAACACACAGCTTCTGTG GAAAAATCCC AGGTTGGTTT CATCGACTAC ATTGTCCATC 1860 CATTGTGGGAGACATGGGCA GATTTGGTAC AGCCTGATGC TCAGGACATT CTCGATACCT 1920 TAGAAGATAACAGGAACTGG TATCAGAGCA TGATACCTCA AAGTCCCTCA CCACCACTGG 1980 ACGAGCAGAACAGGGACTGC CAGGGTCTGA TGGAGAAGTT TCAGTTTGAA CTGACTCTCG 2040 ATGAGGAAGATTCTGAAGGA CCTGAGAAGG AGGGAGAGGG ACACAGCTAT TTCAGCAGCA 2100 CAAAGACGCTTTGTGTGATT GATCCAGAAA ACAGAGATTC CCTGGGAGAG ACTGACATAG 2160 ACATTGCAACAGAAGACAAG TCCCCCGTGG ATACATAATC CCCCTCTCCC TGTGGAGATG 2220 AACATTCTATCCTTGATGAG CATGCCAGCT ATGTGGTAGG GCCAGCCCAC CATGGGGGCC 2280 AAGACCTGCACAGGACAAGG GCCACCTGGC CTTTCAGTTA CTTGAGTTTG GAGTCAGAAA 2340 GCAAGACCAGGAAGCAAATA GCAGCTCAGG AAATCCCACG GTTGACTTGC CTTGATGGCA 2400 AGCTTGGTGGAGAGGACTGA AGCTGTTGCT GGGGGCCGAT TCTGATCAAG ACACATGGCT 2460 TGTAAATGGAAGACACAACA CTGAGAGATC ATTCTGCTCT AAGTTTCGGG AACTTATCCC 2520 CGACAGTGACTGAACTCACT GACTAATAAC TTCC 2554 2246 base pairs nucleic acid doublelinear cDNA 2 CATTTATGCA GATGAGCTTA TAAGAGACCG TTCCCTCCGC CTTCTTCCTCAGAGGAAGTT 60 TCTTGGTAGA TCACCGACAC CTCATCCAGG CGGGGGGTTG GGGGGAAACTTGGCACCAGC 120 CATCCCAGGC AGAGCACCAC TGTGATTTGT TCTCCTGGTG GAGAGAGCTGGAAGGAAGGA 180 GCCAGCGTCC AAATAATGAA GGAGCACGGG GGCACCTTCA GTAGCACCGGAATCAGCGGT 240 GGTACGGGTG ACTCTGCTAT GGACAGCCTG CAGCCGCTCC AGCCTAACTACATGCCTGTG 300 TGTTTGTTTG CAGAAGAATC TTATCAAAAA TTAGCAATGG AAACGCTGGAGGAATTAGAC 360 TGGTGTTTAG ACCAGCTAGA GACCATACAG ACCTACCGGT CTGTCAGTGAGATGGCTTCT 420 AACAAGTTCA AAAGAATGCT GAACCGGGAG CTGACACACC TCTCAGAGATGAGCCGATCA 480 GGGAACCAGG TGTCTGAATA CATTTCAAAT ACTTTCTTAG ACAAGCAGAATGATGTGGAG 540 ATCCCATCTC CTACCCAGAA AGACAGGGAG AAAAAGAAAA AGCAGCAGCTCATGACCCAG 600 ATAAGTGGAG TGAAGAAATT AATGCATAGT TCAAGCCTAA ACAATACAAGCATCTCACGC 660 TTTGGAGTCA ACACTGAAAA TGAAGATCAC CTGGCCAAGG AGCTGGAAGACCTGAACAAA 720 TGGGGTCTTA ACATCTTTAA TGTGGCTGGA TATTCTCACA ATAGACCCCTAACATGCATC 780 ATGTATGCTA TATTCCAGGA AAGAGACCTC CTAAAGACAT TCAGAATCTCATCTGACACA 840 TTTATAACCT ACATGATGAC TTTAGAAGAC CATTACCATT CTGACGTGGCATATCACAAC 900 AGCCTGCACG CTGCTGATGT AGCCCAGTCG ACCCATGTTC TCCTTTCTACACCAGCATTA 960 GACGCTGTCT TCACAGATTT GGAAATCCTG GCTGCCATTT TTGCAGCTGCCATCCATGAC 1020 GTTGATCATC CTGGAGTCTC CAATCAGTTT CTCATCAACA CAAATTCAGAACTTGCTTTG 1080 ATGTATAATG ATGAATCTGT GTTGGAAAAT CATCACCTTG CTGTGGGTTTCAAACTGCTG 1140 CAAGGAGAAC ACTGTGACAT CTTCATGAAT CTCACCAAGA AGCAGCGTCAGACACTCAGG 1200 AAGATGGTTA TTGACATGGT GTTAGCAACT GATATGTCTA AACATATGAGCCTGCTGGCA 1260 GACCTGAAGA CAATGGTAGA AACGAAGAAA GTTACAAGTT CAGGCGTTCTTCTCCTAGAC 1320 AACTATACCG ATCGCATTCA GGTCCTTCGC AACATGGTAC ACTGTGCAGACCTGAGCAAC 1380 CCCACCAAGT CCTTGGAATT GTATCGGCAA TGGACAGACC GCCTCATGGAGGAATTTTTC 1440 CAGCAGGGAG ACAAAGAGCG GGAGAGGGGA ATGGAAATTA GCCCAATGTGTGATAAACAC 1500 ACAGCTTCTG TGGAAAAATC CCAGGTTGGT TTCATCGACT ACATTGTCCATCCATTGTGG 1560 GAGACATGGG CAGATTTGGT ACAGCCTGAT GCTCAGGACA TTCTCGATACCTTAGAAGAT 1620 AACAGGAACT GGTATCAGAG CATGATACCT CAAAGTCCCT CACCACCACTGGACGAGCAG 1680 AACAGGGACT GCCAGGGTCT GATGGAGAAG TTTCAGTTTG AACTGACTCTCGATGAGGAA 1740 GATTCTGAAG GACCTGAGAA GGAGGGAGAG GGACACAGCT ATTTCAGCAGCACAAAGACG 1800 CTTTGTGTGA TTGATCCAGA AAACAGAGAT TCCCTGGGAG AGACTGACATAGACATTGCA 1860 ACAGAAGACA AGTCCCCCGT GGATACATAA TCCCCCTCTC CCTGTGGAGATGAACATTCT 1920 ATCCTTGATG AGCATGCCAG CTATGTGGTA GGGCCAGCCC ACCATGGGGGCCAAGACCTG 1980 CACAGGACAA GGGCCACCTG GCCTTTCAGT TACTTGAGTT TGGAGTCAGAAAGCAAGACC 2040 AGGAAGCAAA TAGCAGCTCA GGAAATCCCA CGGTTGACTT GCCTTGATGGCAAGCTTGGT 2100 GGAGAGGACT GAAGCTGTTG CTGGGGGCCG ATTCTGATCA AGACACATGGCTTGTAAATG 2160 GAAGACACAA CACTGAGAGA TCATTCTGCT CTAAGTTTCG GGAACTTATCCCCGACAGTG 2220 ACTGAACTCA CTGACTAATA ACTTCC 2246 2045 base pairsnucleic acid double linear cDNA 3 GTGGAAGCAA ACAGCGGAGG CAAGGGGTTGTTTCGGACAC ACTAGAGAGT AAGTCAGAGA 60 ATCTTCGTGT TGAGGCAGCA TTGCAAAATTGAAGATGAAG AAAGGAAGGA AGAAGAATCT 120 TATCAAAAAT TAGCAATGGA AACGCTGGAGGAATTAGACT GGTGTTTAGA CCAGCTAGAG 180 ACCATACAGA CCTACCGGTC TGTCAGTGAGATGGCTTCTA ACAAGTTCAA AAGAATGCTG 240 AACCGGGAGC TGACACACCT CTCAGAGATGAGCCGATCAG GGAACCAGGT GTCTGAATAC 300 ATTTCAAATA CTTTCTTAGA CAAGCAGAATGATGTGGAGA TCCCATCTCC TACCCAGAAA 360 GACAGGGAGA AAAAGAAAAA GCAGCAGCTCATGACCCAGA TAAGTGGAGT GAAGAAATTA 420 ATGCATAGTT CAAGCCTAAA CAATACAAGCATCTCACGCT TTGGAGTCAA CACTGAAAAT 480 GAAGATCACC TGGCCAAGGA GCTGGAAGACCTGAACAAAT GGGGTCTTAA CATCTTTAAT 540 GTGGCTGGAT ATTCTCACAA TAGACCCCTAACATGCATCA TGTATGCTAT ATTCCAGGAA 600 AGAGACCTCC TAAAGACATT CAGAATCTCATCTGACACAT TTATAACCTA CATGATGACT 660 TTAGAAGACC ATTACCATTC TGACGTGGCATATCACAACA GCCTGCACGC TGCTGATGTA 720 GCCCAGTCGA CCCATGTTCT CCTTTCTACACCAGCATTAG ACGCTGTCTT CACAGATTTG 780 GAAATCCTGG CTGCCATTTT TGCAGCTGCCATCCATGACG TTGATCATCC TGGAGTCTCC 840 AATCAGTTTC TCATCAACAC AAATTCAGAACTTGCTTTGA TGTATAATGA TGAATCTGTG 900 TTGGAAAATC ATCACCTTGC TGTGGGTTTCAAACTGCTGC AAGGAGAACA CTGTGACATC 960 TTCATGAATC TCACCAAGAA GCAGCGTCAGACACTCAGGA AGATGGTTAT TGACATGGTG 1020 TTAGCAACTG ATATGTCTAA ACATATGAGCCTGCTGGCAG ACCTGAAGAC AATGGTAGAA 1080 ACGAAGAAAG TTACAAGTTC AGGCGTTCTTCTCCTAGACA ACTATACCGA TCGCATTCAG 1140 GTCCTTCGCA ACATGGTACA CTGTGCAGACCTGAGCAACC CCACCAAGTC CTTGGAATTG 1200 TATCGGCAAT GGACAGACCG CCTCATGGAGGAATTTTTCC AGCAGGGAGA CAAAGAGCGG 1260 GAGAGGGGAA TGGAAATTAG CCCAATGTGTGATAAACACA CAGCTTCTGT GGAAAAATCC 1320 CAGGTTGGTT TCATCGACTA CATTGTCCATCCATTGTGGG AGACATGGGC AGATTTGGTA 1380 CAGCCTGATG CTCAGGACAT TCTCGATACCTTAGAAGATA ACAGGAACTG GTATCAGAGC 1440 ATGATACCTC AAAGTCCCTC ACCACCACTGGACGAGCAGA ACAGGGACTG CCAGGGTCTG 1500 ATGGAGAAGT TTCAGTTTGA ACTGACTCTCGATGAGGAAG ATTCTGAAGG ACCTGAGAAG 1560 GAGGGAGAGG GACACAGCTA TTTCAGCAGCACAAAGACGC TTTGTGTGAT TGATCCAGAA 1620 AACAGAGATT CCCTGGGAGA GACTGACATAGACATTGCAA CAGAAGACAA GTCCCCCGTG 1680 GATACATAAT CCCCCTCTCC CTGTGGAGATGAACATTCTA TCCTTGATGA GCATGCCAGC 1740 TATGTGGTAG GGCCAGCCCA CCATGGGGGCCAAGACCTGC ACAGGACAAG GGCCACCTGG 1800 CCTTTCAGTT ACTTGAGTTT GGAGTCAGAAAGCAAGACCA GGAAGCAAAT AGCAGCTCAG 1860 GAAATCCCAC GGTTGACTTG CCTTGATGGCAAGCTTGGTG GAGAGGACTG AAGCTGTTGC 1920 TGGGGGCCGA TTCTGATCAA GACACATGGCTTGTAAATGG AAGACACAAC ACTGAGAGAT 1980 CATTCTGCTC TAAGTTTCGG GAACTTATCCCCGACAGTGA CTGAACTCAC TGACTAATAA 2040 CTTCC 2045 721 amino acids aminoacid single linear peptide 4 Met Thr Ala Lys Asp Ser Ser Lys Glu Leu ThrAla Ser Glu Pro Glu 1 5 10 15 Val Cys Ile Lys Thr Phe Lys Glu Gln MetHis Leu Glu Leu Glu Leu 20 25 30 Pro Arg Leu Pro Gly Asn Arg Pro Thr SerPro Lys Ile Ser Pro Arg 35 40 45 Ser Ser Pro Arg Asn Ser Pro Cys Phe PheArg Lys Leu Leu Val Asn 50 55 60 Lys Ser Ile Arg Gln Arg Arg Arg Phe ThrVal Ala His Thr Cys Phe 65 70 75 80 Asp Val Glu Asn Gly Pro Ser Pro GlyArg Ser Pro Leu Asp Pro Gln 85 90 95 Ala Ser Ser Ser Ala Gly Leu Val LeuHis Ala Thr Phe Pro Gly His 100 105 110 Ser Gln Arg Arg Glu Ser Phe LeuTyr Arg Ser Asp Ser Asp Tyr Asp 115 120 125 Leu Ser Pro Lys Ala Met SerArg Asn Ser Ser Leu Pro Ser Glu Gln 130 135 140 His Gly Asp Asp Leu IleVal Thr Pro Phe Ala Gln Val Leu Ala Ser 145 150 155 160 Leu Arg Ser ValArg Asn Asn Phe Thr Ile Leu Thr Asn Leu His Gly 165 170 175 Thr Ser AsnLys Arg Ser Pro Ala Ala Ser Gln Pro Pro Val Ser Arg 180 185 190 Val AsnPro Gln Glu Glu Ser Tyr Gln Lys Leu Ala Met Glu Thr Leu 195 200 205 GluGlu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile Gln Thr Tyr 210 215 220Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg Met Leu Asn 225 230235 240 Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val245 250 255 Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln Asn Asp ValGlu 260 265 270 Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys Lys LysGln Gln 275 280 285 Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met HisSer Ser Ser 290 295 300 Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val AsnThr Glu Asn Glu 305 310 315 320 Asp His Leu Ala Lys Glu Leu Glu Asp LeuAsn Lys Trp Gly Leu Asn 325 330 335 Ile Phe Asn Val Ala Gly Tyr Ser HisAsn Arg Pro Leu Thr Cys Ile 340 345 350 Met Tyr Ala Ile Phe Gln Glu ArgAsp Leu Leu Lys Thr Phe Arg Ile 355 360 365 Ser Ser Asp Thr Phe Ile ThrTyr Met Met Thr Leu Glu Asp His Tyr 370 375 380 His Ser Asp Val Ala TyrHis Asn Ser Leu His Ala Ala Asp Val Ala 385 390 395 400 Gln Ser Thr HisVal Leu Leu Ser Thr Pro Ala Leu Asp Ala Val Phe 405 410 415 Thr Asp LeuGlu Ile Leu Ala Ala Ile Phe Ala Ala Ala Ile His Asp 420 425 430 Val AspHis Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser 435 440 445 GluLeu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu Asn His His 450 455 460Leu Ala Val Gly Phe Lys Leu Leu Gln Gly Glu His Cys Asp Ile Phe 465 470475 480 Met Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg Lys Met Val Ile485 490 495 Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Ser Leu LeuAla 500 505 510 Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser SerGly Val 515 520 525 Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val LeuArg Asn Met 530 535 540 Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys SerLeu Glu Leu Tyr 545 550 555 560 Arg Gln Trp Thr Asp Arg Leu Met Glu GluPhe Phe Gln Gln Gly Asp 565 570 575 Lys Glu Arg Glu Arg Gly Met Glu IleSer Pro Met Cys Asp Lys His 580 585 590 Thr Ala Ser Val Glu Lys Ser GlnVal Gly Phe Ile Asp Tyr Ile Val 595 600 605 His Pro Leu Trp Glu Thr TrpAla Asp Leu Val Gln Pro Asp Ala Gln 610 615 620 Asp Ile Leu Asp Thr LeuGlu Asp Asn Arg Asn Trp Tyr Gln Ser Met 625 630 635 640 Ile Pro Gln SerPro Ser Pro Pro Leu Asp Glu Gln Asn Arg Asp Cys 645 650 655 Gln Gly LeuMet Glu Lys Phe Gln Phe Glu Leu Thr Leu Asp Glu Glu 660 665 670 Asp SerGlu Gly Pro Glu Lys Glu Gly Glu Gly His Ser Tyr Phe Ser 675 680 685 SerThr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn Arg Asp Ser Leu 690 695 700Gly Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys Ser Pro Val Asp 705 710715 720 Thr 564 amino acids amino acid single linear peptide 5 Met LysGlu His Gly Gly Thr Phe Ser Ser Thr Gly Ile Ser Gly Gly 1 5 10 15 SerGly Asp Ser Ala Met Asp Ser Leu Gln Pro Leu Gln Pro Asn Tyr 20 25 30 MetPro Val Cys Leu Phe Ala Glu Glu Ser Tyr Gln Lys Leu Ala Met 35 40 45 GluThr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile 50 55 60 GlnThr Tyr Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg 65 70 75 80Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly 85 90 95Asn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln Asn 100 105110 Asp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys Lys 115120 125 Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met His130 135 140 Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val AsnThr 145 150 155 160 Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu Asp LeuAsn Lys Trp 165 170 175 Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser HisAsn Arg Pro Leu 180 185 190 Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu ArgAsp Leu Leu Lys Thr 195 200 205 Phe Arg Ile Ser Ser Asp Thr Phe Ile ThrTyr Met Met Thr Leu Glu 210 215 220 Asp His Tyr His Ser Asp Val Ala TyrHis Asn Ser Leu His Ala Ala 225 230 235 240 Asp Val Ala Gln Ser Thr HisVal Leu Leu Ser Thr Pro Ala Leu Asp 245 250 255 Ala Val Phe Thr Asp LeuGlu Ile Leu Ala Ala Ile Phe Ala Ala Ala 260 265 270 Ile His Asp Val AspHis Pro Gly Val Ser Asn Gln Phe Leu Ile Asn 275 280 285 Thr Asn Ser GluLeu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu 290 295 300 Asn His HisLeu Ala Val Gly Phe Lys Leu Leu Gln Gly Glu His Cys 305 310 315 320 AspIle Phe Met Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg Lys 325 330 335Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Ser 340 345350 Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser 355360 365 Ser Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val Leu370 375 380 Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys SerLeu 385 390 395 400 Glu Leu Tyr Arg Gln Trp Thr Asp Arg Leu Met Glu GluPhe Phe Gln 405 410 415 Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu IleSer Pro Met Cys 420 425 430 Asp Lys His Thr Ala Ser Val Glu Lys Ser GlnVal Gly Phe Ile Asp 435 440 445 Tyr Ile Val His Pro Leu Trp Glu Thr TrpAla Asp Leu Val Gln Pro 450 455 460 Asp Ala Gln Asp Ile Leu Asp Thr LeuGlu Asp Asn Arg Asn Trp Tyr 465 470 475 480 Gln Ser Met Ile Pro Gln SerPro Ser Pro Pro Leu Asp Glu Gln Asn 485 490 495 Arg Asp Cys Gln Gly LeuMet Glu Lys Phe Gln Phe Glu Leu Thr Leu 500 505 510 Asp Glu Glu Asp SerGlu Gly Pro Glu Lys Glu Gly Glu Gly His Ser 515 520 525 Tyr Phe Ser SerThr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn Arg 530 535 540 Asp Ser LeuGly Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys Ser 545 550 555 560 ProVal Asp Thr 517 amino acids amino acid single linear peptide 6 Met GluThr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr 1 5 10 15 IleGln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys 20 25 30 ArgMet Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser 35 40 45 GlyAsn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln 50 55 60 AsnAsp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys 65 70 75 80Lys Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met 85 90 95His Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn 100 105110 Thr Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys 115120 125 Trp Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro130 135 140 Leu Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu LeuLys 145 150 155 160 Thr Phe Arg Ile Ser Ser Asp Thr Phe Ile Thr Tyr MetMet Thr Leu 165 170 175 Glu Asp His Tyr His Ser Asp Val Ala Tyr His AsnSer Leu His Ala 180 185 190 Ala Asp Val Ala Gln Ser Thr His Val Leu LeuSer Thr Pro Ala Leu 195 200 205 Asp Ala Val Phe Thr Asp Leu Glu Ile LeuAla Ala Ile Phe Ala Ala 210 215 220 Ala Ile His Asp Val Asp His Pro GlyVal Ser Asn Gln Phe Leu Ile 225 230 235 240 Asn Thr Asn Ser Glu Leu AlaLeu Met Tyr Asn Asp Glu Ser Val Leu 245 250 255 Glu Asn His His Leu AlaVal Gly Phe Lys Leu Leu Gln Gly Glu His 260 265 270 Cys Asp Ile Phe MetAsn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg 275 280 285 Lys Met Val IleAsp Met Val Leu Ala Thr Asp Met Ser Lys His Met 290 295 300 Ser Leu LeuAla Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr 305 310 315 320 SerSer Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val 325 330 335Leu Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser 340 345350 Leu Glu Leu Tyr Arg Gln Trp Thr Asp Arg Leu Met Glu Glu Phe Phe 355360 365 Gln Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met370 375 380 Cys Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly PheIle 385 390 395 400 Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala AspLeu Val Gln 405 410 415 Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu AspAsn Arg Asn Trp 420 425 430 Tyr Gln Ser Met Ile Pro Gln Ser Pro Ser ProPro Leu Asp Glu Gln 435 440 445 Asn Arg Asp Cys Gln Gly Leu Met Glu LysPhe Gln Phe Glu Leu Thr 450 455 460 Leu Asp Glu Glu Asp Ser Glu Gly ProGlu Lys Glu Gly Glu Gly His 465 470 475 480 Ser Tyr Phe Ser Ser Thr LysThr Leu Cys Val Ile Asp Pro Glu Asn 485 490 495 Arg Asp Ser Leu Gly GluThr Asp Ile Asp Ile Ala Thr Glu Asp Lys 500 505 510 Ser Pro Val Asp Thr515

1. An DNA segment comprising the DNA sequence of: (a) SEQUENCE ID NO. 1or an alleleic variation thereof; (b) SEQUENCE ID NO. 2 or an alleleicvariation thereof; or (c) SEQUENCE ID NO. 3 or an alleleic variationthereof.
 2. A DNA segment that is a subset of and functionallyequivalent to a DNA segment according to claim
 1. 3. A substantiallypurified polypeptide comprising the amino acid sequence of SEQUENCE IDNO. 4, SEQUENCE ID NO. 5 or SEQUENCE ID NO.
 6. 4. A polypeptide that isa subset of and that is functionally equivalent to a polypeptideaccording to claim
 3. 5. A polypeptide produced by expression in a hostcell into which has been incorporated the DNA sequence of: (a) SEQUENCEID NO. 1 or an alleleic variation thereof; (b) SEQUENCE ID NO. 2 or analleleic variation thereof; or (c) SEQUENCE ID NO. 3 or an alleleicvariation thereof.
 6. Recombinant DNA comprising a DNA segment accordingto claim
 1. 7. Recombinant DNA according to claim 6 comprising the DNAsequence of SEQUENCE ID NO. 1 or an alleleic variation thereof. 8.Recombinant DNA according to claim 6 comprising the DNA sequence ofSEQUENCE ID NO. 2 or an alleleic variation thereof.
 9. Recombinant DNAaccording to claim 6 comprising the DNA sequence of SEQUENCE ID NO. 3 oran alleleic variation thereof.
 10. A DNA vector comprising recombinantDNA according to claim
 6. 11. A DNA vector according to claim 8, whereinsaid vector is pcDNA1-amp.
 12. A host cell into which has beenincorporated recombinant DNA according to claim
 6. 13. An isolated DNAsegment comprising the genomic promoter region that regulatestranscription or translation of: (a) the DNA sequence of SEQUENCE ID NO.1 or an alleleic variation thereof; or (b) the DNA sequence of SEQUENCEID NO. 2 or an alleleic variation thereof; or (c) the DNA sequence ofSEQUENCE ID NO. 3 or an alleleic variation thereof.
 14. A method ofdetecting the presence of hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3 in humancells comprising: (a) performing a reverse transcriptase-polymerasechain reaction on total RNA from such cells using a pair of polymerasechain reaction primers that are specific for, respectively, hPDE IV-B1,hPDE IV-B2 or hPDE IV-B3, as determined from, respectively: (i) the DNAsequence of SEQUENCE ID NO. 1 or an alleleic variation thereof; or (ii)the DNA sequence of SEQUENCE ID NO. 2 or an alleleic variation thereof;or (iii) the DNA sequence of SEQUENCE ID NO. 3 or an alleleic variationthereof; and (b) assaying the appearance of an appropriately sized PCRfragment by agarose gel electrophoresis.
 15. A method of identifyingcompounds or other substances that inhibit or modify the activity ofhPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, comprising measuring the activityof, respectively, hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, in (a) a cellline into which has been incorporated recombinant DNA according to claim6, or (b) a cell line that naturally selectively expresses hPDE IV-B1,hPDE IV-b2 or hPDE IV-B3, as determined by the method of claim
 14. 16.An isolated DNA segment comprising a DNA sequence that is a subset ofthe DNA sequence of SEQUENCE ID NO. 1, or an alleleic variation thereof,and that is capable of hybridizing to the DNA sequence of SEQUENCE IDNO. 1, or an alleleic variation thereof, when used as a probe, oramplifying all or part of such sequence when used as a polymerase chainreaction primer.
 17. An isolated DNA segment comprising a DNA sequencethat is a subset of the DNA sequence of SEQUENCE ID NO. 2, or analleleic variation thereof, and that is capable of hybridizing to theDNA sequence of SEQUENCE ID NO. 2, or an alleleic variation thereof,when used as a probe, or amplifying all or part of such sequence whenused as a polymerase chain reaction primer.
 18. An isolated DNA segmentcomprising a DNA sequence that is a subset of the DNA sequence ofSEQUENCE ID NO. 3, or an alleleic variation thereof, and that is capableof hybridizing to the DNA sequence of SEQUENCE ID NO. 3, or an alleleicvariation thereof, when used as a probe, or amplifying all or part ofsuch sequence when used as a polymerase chain reaction primer.